This invention relates to a novel DNA sequence for a heparin-binding neurotrophic factor (HBNF). The sequence of the invention encodes a protein which is capable of inducing nerve cell growth and differentiation, as well as nerve cell maintenance and repair, both in vivo and in vitro.
The protein in question is normally produced in the human brain and homologous forms exist in a number of different species. The proteins have also been previously referred to as a heparin-binding brain mitogens (HBBMs). Although the purified proteins are known, the only available source of the proteins has been from brain tissue extracts. The procedure for isolation from brain tissue is laborious and yields relatively small quantities of HBNF.
The gene encoding the human HBNF has now been isolated from a cDNA library obtained from newborn human brain stem RNA. It is a 411 nucleotide sequence predicting a protein having 136 amino acids with a molecular weight of about 15 KD. The gene has been sequenced and expressed in E. coli, and the protein so produced retains the neurotrophic activity of the native HBNF.
In recent years a number of relatively small polypeptides, known as growth factors, have been identified and isolated. The term xe2x80x9cgrowth factorsxe2x80x9d refers to a class of signalling substances which affect the growth and differentiation of certain types of animals; this effect can be seen both in the animal and in tissue culture. A given growth factor may have an effect on more than one type of cell.
Many of the better known growth factors have significant neurotrophic activity, i.e., they are capable of maintaining or stimulating growth of nerve cells. The earliest discovery of such a neurotrophic factor was nerve growth factor (NGF; Gospodarowicz, J. Biol. Chem. 250: 2515-2520, 19757. Similar growth factors which are in the same family as NGF are brain-derived neurotrophic factor (BDNF; Leibrock et al., Nature 341: 149-153, 1989) and neurotrophic factor xe2x80x943 (NT-3; Maisonpierre et al., Science 247: 1446-1451, 1990). Additional growth factors include ciliary neurotrophic factor (CNTF; Lin et al, Science 246: 1023-1025, 1980, IGF-II (Mill et al, PNAS USA 82: 7126-7130, 1985), activin (Schubert et al., Nature 344: 868-870, 1990) and purpurin (Berman et al., Cell 51: 135-142, 1987).
A number of other known factors fall into a superfamily related to fibroblast growth factor (FGF); this includes basic FGF(bFGF), Esch et al., PNAS USA 81: 5364-5368; PNAS USA 82:6507-6511), acidic FGF (aFGF), (Bohlen et al., EMBO. J. 4 1951-1956, 1985; Gimenez-Gallego et al., Science 230: 1385-1288, 1985), as well as products of the oncogenes intxe2x80x942 (Dickens and Peters, Nature 326:833, 1984), hst/KS (Delli Bovi et al., Cell 50: 729-737, 1987) FGF-5 (Zhan et al., Mol. Cell Biol. 8:3487-3495, 1988), FGF-6 (Marics et al., Oncogene 4: 335-340, 1989) and KGF (Finch et al., Science 245: 752-755, 1989). These are all (except KGF) mitogens for vascular endothelial cells, and all also bind strongly to heparin. Other heparin-binding growth factors, such as VEGF/VPF, are also known (Keck et al, Science 246: 1309-1312, 1989). These heparin-binding growth factors are also frequently isolated from brain tissue and may play a significant role in the growth and development of brain cells.
A previously unknown heparin binding protein was described in EP 326 075, and was referred to therein as HBBM; it was disclosed as a brain mitogen as well as a tissue formation, maintenance and repair factor, particularly for neural tissue. It is also structurally unrelated to any of the aforementioned growth factors, although it appears to be structurally related to a protein the gene of which was previously referred to as MK (Kadomatsu et al., Biochem Biophys. Res. Comm. 151: 1312-1318, 1288) and a human form of the MK protein. The homology between HBNF and MK genes and proteins is very high, and they are assumed to constitute a new family of neurotrophic factors. The xe2x80x9cHBBMxe2x80x9d protein is the xe2x80x9cHBNFxe2x80x9d protein of the present invention. However, as indicated in the aforementioned European Application, it has previously been necessary to isolate the protein directly from brain tissue by a procedure involving several chromatographic steps, as neither the complete protein sequence nor the gene sequence was previously known.
More recently, HBNF proteins have been isolated from both rat (Rauvala, EMBO J. 8: 2933-2941, 1989; Huber et al Neurochem Res. 15: 435-439, 1990), and cow (Milner et al., Biochem. Biophys. Res. Comm. 165: 1096-1103, 1989; Huber et al Neurochem Res. 15:435-439, 1990), and the amino terminal sequences have been determined. Similarly, the N-terminal amino acid sequences of the human and chicken proteins have been determined (EP 326 075; Huber et al, Neurochem. Res. 15: 435-439, 1990). Moreover, no determination of the DNA sequence of the HBNF has previously been achieved. The present invention now provides an entire gene sequence for human HBNF, as well as cloning vectors and host cells capable of expressing the gene and producing pure HRNF protein. The invention also provides both in vitro and in vivo methods of promoting nerve cell growth, repair and maintenance.